Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA

Abstract

Environmental DNA (eDNA) is increasingly used to monitor aquatic macrofauna. Typically, short mitochondrial DNA fragments are targeted because these should be relatively more abundant in the environment as longer fragments will break into smaller fragments over time. However, longer fragments may permit more flexible primer design and increase taxonomic resolution for eDNA metabarcoding analyses, and recent studies have shown that long mitochondrial eDNA fragments can be extracted from environmental water samples. Nuclear eDNA fragments have also been proposed as targets, but little is known about their persistence in the aquatic environment. Here we measure the abundance of mitochondrial eDNA fragments of different lengths and of short nuclear eDNA fragments, originating from captive fish in experimental tanks, and we test whether longer mitochondrial and short nuclear fragments decay faster than short mitochondrial fragments following fish removal. We show that when fish are present, shorter mitochondrial fragments are more abundant in water samples than both longer mitochondrial fragments and short nuclear eDNA fragments. However, the rate of decay following fish removal was similar for all fragment types, suggesting that the differences in abundance resulted from differences in the rates at which different fragment types were produced rather than differences in their decay rates.

Effect of water temperature and fish biomass on environmental DNA shedding, degradation, and size distribution

“…It is also the first to show the water temperature‐dependent degradation of marine fish eDNA. As moderately higher temperatures (<50°C) stimulate microbial metabolism and exonuclease activity (Corinaldesi, Beolchini, & Dell'Anno, ; Poté, Ackermann, & Wildi, ), and high fish density can lead to the increase in microbial activity (Barnes et al, ; Bylemans et al, ), these results are likely to support the hypothesis that the activity and abundance of microbes and extracellular nucleases significantly affect eDNA degradation (Barnes & Turner, ; Levy‐Booth et al, ; Nielsen, Johnsen, Bensasson, & Daffonchio, ).…”

“…The main factors associated with eDNA shedding are (1) the number and the biomass of organisms (Klymus, Richter, Chapman, & Paukert, 2015;Takahara, Minamoto, Yamanaka, Doi, & Kawabata, 2012); (2) the developmental stage of the organisms (Maruyama, Nakamura, Yamanaka, Kondoh, & Minamoto, 2014); (3) the behavior of organisms (Dunn, Priestley, Herraiz, Arnold, & Savolainen, 2017); and (4) the stress against organisms (Bylemans, Furlan, Gleeson, Hardy, & Duncan, 2018;Pilliod et al, 2014). In addition, considering that feed intake increased the eDNA shedding (Klymus et al, 2015), eDNA shedding rate is likely to depend on (5) the metabolism and physiological activity of the organisms.…”

Toward an ecoregion scale evaluation of eDNA metabarcoding primers: A case study for the freshwater fish biodiversity of the Murray–Darling Basin (Australia)

“…Otherwise, the shorter barcoding region amplified by the Teleo primers could also increase the detection of fish taxa due to an increased ability to recover highly degraded eDNA. Although recent studies have suggested that the aquatic environment may preserve eDNA relatively well (Bylemans et al., ; Piggott, ), more research is needed to evaluate the effect of barcode length on eDNA metabarcoding surveys. Overall, the results show that even within an ecoregion the performance of eDNA metabarcoding primers may differ depending on the local biodiversity.…”

“…First, a positive relationship exists between the length of the internally amplified barcode and its taxonomic resolution power (Coissac et al., ; Meusnier et al., ) but the ability to recover DNA from environmental samples can be negatively impacted by the size of the DNA fragments (Deagle, Eveson, & Jarman, ; Jo et al., ). However, a number of recent studies have shown that this may be less problematic for eDNA derived from water samples (Bylemans, Furlan, Gleeson, Hardy, & Duncan, ; Deiner et al., ; Piggott, ). Second, reducing primer‐template mismatches can minimize biases arising from the PCR amplification but may inadvertently decrease the specificity of the primers to the taxonomic group of interest as these primers are more likely to bind to highly conserved regions (Pinol, Mir, Gomez‐Polo, & Agusti, ).…”

Development and application of eDNA-based tools for the conservation of white-clawed crayfish

“…In the case of species-specific eDNA assays, the design and validation of the assay represents a critical first 47 step (Geerts et al, 2018). During assay design, it is fundamental to ensure a high target specificity 48 (Bylemans et al, 2018) by selecting suitable amplicon lengths, in-silico simulations and testing against 49 amplification of non-target DNA. In-vitro laboratory validation should then ascertain that the assay 50 complies with established guidelines (Bustin et al, 2009) and that limits of detection (LOD) and 51 quantification (LOQ) are established.…”